Cyanobacteria, also known as blue-green algae, are photosynthetic bacteria widespread in marine and freshwater environments. Cyanobacteria have simple growth requirements and grow to high densities by using light, carbon dioxide, and other inorganic nutrients for growth. They are useful hosts for the production of a wide range of compounds of interest including biofuels and other commodity chemicals, for example.
A barrier to using cyanobacteria for the production of compounds of interest is that efficient introduction of engineered vectors in cyanobacteria is hampered, or completely prohibited, due to the presence of restriction nucleases, see Thorsten H. et. al., Ch. 24, Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions, In: Methods in Enzymology, Academic Press, 2011, Vol. 497, pp. 539-579, These nucleases can significantly decrease or completely prevent the uptake of exogenous DNA into cyanobacteria) cells, see for example Ruffing AM. et. al., Biotech. Bioeng., 2012, Vol. 109, pp. 2190-2199. Enzymes exhibiting nuclease activity are usually either endonucleolytic (an endonuclease) or exonucleolytic (an exonuclease) and may be either sugar specific or non-sugar specific. A catalog of restriction enzymes and their recognition sites is compiled and maintained by New England Biolabs in Ipswich, Massachusetts and can be accessed at http: //rebase.neb.com/ (REBASE).
In a prophetic embodiment, ROligos could be labeled with fluorescent dyes, radiolabels, antigens or other labels. The labels could be incorporated through post-labeling techniques or could be incorporated using pre-labeled nucleotides through polynucleotide synthetic techniques well known in the art, see for example oligonucleotide synthetic services available through Iba life sciences in Olivette, MO at http://www.lba-lifesciences.com/Service Custom oliqos.html and Shibata A., Molecules 2012 Feb. 29; 17(3)(42446-63 which discloses various techniques and references to techniques well known in the art of oligonucleotide labeling,
Various restriction endonucleases exhibit restriction activity to varying degrees in various buffer conditions. Buffer conditions conunonly used for known restriction enzymes are mostly covered across four different buffers available through New England Biolabs, P1, P2, P3 and P4 buffers. Their compositions can be found at REBASE and are well known to those having skill in the art.
Cyanobacteria often contain one or more sequence-specific restriction endonucleases. These nucleases are almost always associated with their cognate methyltransferase. The methyltransferase methylates the host chromosomal and plasmid DNA to provide protection against its associated restriction enzyme. Endogenous cyanobacterial DNA is thus protected against specific nuclease activity by specific methylation, but foreign DNA is not methylated and is therefore targeted for destruction by the cyanobacterial nucleases, see Soper, B W et. al, J. Bacteriology 1994, 176(17):556-5570 and Elhai, J, et. al, J. Bacteriology 1997, 179(6): 1998-2005.